Purification process

ABSTRACT

Object of the invention is a process for the chromatographic purification of cyclosporin A from a crude product containing cyclosporin complex by using a column filled with silica gel and by the application of multistep chromatography with a column filled with normal phase silica gel and by a solvent mixture containing toluene as the major component.

[0001] The object of the invention is a process for the chromatographicpurification of cyclosporin A from a crude product containingcyclosporin complex by using a normal phase—column filled with silicagel or an equivalent matrix and by the application of multistepchromatography with a column filled with normal phase silica gel or anequivalent matrix and by a solvent or solvent mixture containing tolueneas the major component.

[0002] Cyclosporins are cyclic undecapeptides being N-methylated atseveral places and most of them having approved pharmacological effects.Twenty five members of this group of compounds have been known so farwhich are designated by letters A to Z. At first cyclosporin A wasseparated among them, which is a natural material and was isolated fromthe culture broth of Tolypocladium inflatum Gams strain (Helv. Chim.Acta 59 1075(1976)). This compound first became known as a slightantifungal antibiotic and later attracted the attention as animmunosuppressive. (J. F. Borel et al: Immunology 32 1017(1977)).Cyclosporin A is mainly used in organ transplantation from other persons(lung, heart, kidney, bone marrow, skin). Pharmacological examinationsproved that it inhibits both the humoral and cellular immune responsesby hindering the proliferation of T-cells and interrupting the synthesisof interleukin-2. Cyclosporin A also has been used for treatingautoimmune and inflammatory diseases, such as in autoimmunehematological diseases, ulcerous colitis, Graves-illness, multiplesclerosis, psoriasis, rheumatic arthritis as well. Further experimentswere done to cure infections caused by protozoa and tumors. Theimportance of cyclosporins is indicated by the fact, that number ofsynthetic related compounds call be prepared by building in differentamino acids and substitutes. (e.g. EP 56782, CH 630062 and EP 29122patents)

[0003] Cyclosporins are prepared by fermentation. Cylindrocarpon LucidumBooth (patent specification No. CH 589,716); Trichoderma polysporumRifai (patent specification No. CH 603,790); Tolypocladium varium(patent specification No. HU 201,577) are used to prepare cyclosporins.At the end of the fermentation, a cyclosporin complex is formed whichmay contain other impurities too (ingredients of culture media,anti-foaming agent, metabolites, etc.) depending on the character of theprocess.

[0004] Generally the product is isolated from the broth by extractionprocesses. This can be done by separating the mycelium from the broth bycentrifugation or filtration, then dissolving the active ingredient fromthe mycelium by methanol or acetone and extracting the filtrate bywater-immiscible solvents. An other known execution method is a processwithout filtration, using whole broth extraction with water-immiscibleorganic solvent. Solvent content of the organic phase is evaporated byvacuum distillation. However during the organic solvent extraction thecompounds having lipid characters are also transferred into the organicphase, which cause difficulties in the further purification. In order toseparate these compounds there are known processes (e.g. Swiss patentspecification No. 589,716 or published German patent application No.2,455,859) where after removing the extracting solvent the residue isdissolved in a mixture of methanol-water and then is extracted severaltimes by the same volume of petroleum ether. Petroleum ether portionsare combined and cyclosporins are recovered from them by a mixture ofmethanol-water. The active substance is transferred by multi-extractionfrom the combined methanol-water phase into ethylene chloride, which isthen washed with water and evaporated to dryness. The crude productprepared by the above method and containing cyclosporins can be purifiedmore efficiently by one of the chromatographic methods.

[0005] According to a method described in U.S. Pat. No. 4,117,118 thecyclosporin mixture is transferred first to a Sephadex LH-20 column andeluted with methanol, then it is eluted successively in an aluminacolumn with a mixture of toluene and ethyl acetate (15%), and in asilica gel column with a mixture of chloroform and methanol (2%).Despite of the repeated chromatography the resulting product is notpure, but it is a mixture of cyclosporin A and B.

[0006] A similar chromatographic process is disclosed among others inthe U.S. Pat. No. 4,215,199, where a rough-cleaning is with a 98:2% v/vmixture of chloroform and methanol on a silica gel column. The eluate isthen evaporated to dryness. The residue is dissolved in methanol and issubjected to chromatography in a Sephadex LH-20 column using methanol aseluent. The eluate fractions are evaporated to dryness, then the residueis dissolved in 98:2% v/v mixture of chloroform and methanol. It issubjected again to a silica gel chromatography. Cyclosporin A appearsfirst in the eluate. This and the consecutive fractions are separated,and the pure components are obtained by evaporating the elutes.

[0007] According to the German Pat. No. DD 298,276 the oily crudeproduct is dissolved in small quantity of chloroform, then subjected tochromatography in an alumina column with chloroform. The fractionscontaining cyclosporin A are evaporated in vacuum, dissolved inchloroform, subjected to a similar column and then eluted by chloroform.Fractions containing the active substance are evaporated in vacuumagain. Hexane is added to the residue and the cyclosporin A iscrystallized. Product is washed by hexane then dried and finallyrecrystallized from a mixture of ether and hexane or acetone.

[0008] According to the Hungarian Pat. No. 201,577 the crude productobtained after evaporation can be cleaned on a silica gel column byelution with a mixture of chloroform and methanol gradually increasingconcentration of methanol. The process is started by pure chloroform andcontinued by increasing in 0.5 vol % steps the methanol in the eluate.Cyclosporin A is eluted by 2 vol %, cyclosporin B by 2.5 vol %,cyclosporin C by 3 vol % methanol containing chloroform from the column.Components are obtained by evaporating the fractions.

[0009] Processes mentioned above describe mainly cyclosporinfermentation procedures, where the first aim of the purification stepsis to identify the product obtained. Thus the product is isolated onlyin a small quantity and only the physical and chemical characteristicsare given without publishing data relating to the purity of the productand the quantities of the impurities.

[0010] Rüieger and Co. /Helv.Chim. Acta 59(4), p. 1075-92 (1976)isolated small quantities of pure cyclosporin A and C by repeatedchromatographies and by other purification steps for identification andstructure analysis. According to this article the crude product receivedfrom the fermentation of Trichoderma polysporum Rifai containing mainlycyclosporins A and C is defatted with methanol and petroleum ether.After evaporation the residue is dissolved in chloroform and issubjected to chromatography by gradient elution with 98.5:1.5 v/vmixture of chloroform and methanol as eluent. Pure crystallinecyclosporin A is obtained by further chromatography. The fractioncontaining cyclosporin A is dissolved in methanol and is subjected tochromatography in a Sephadex LH-20 column by using methanol as eluent.Peak fractions are evaporated, dissolved in toluene and subjected tochromatography in a column packed with aluminum oxide, using toluene aseluent, in the presence of an increasing, concentration of acetic acid.The crystalline product is obtained after the evaporation of fractionsand a treatment with activated carbon in all alcoholic solution.

[0011] A purification process feasible in industrial scale is describedin the U.S. Pat. No. 5,382,655. According to the process the crudeproduct containing different cyclosporin components is subjected to heattreatment prior to chromatography on silica gel column by the mixturesof chloroform-dichloromethane-ethanol or chloroform-ethylacetate-ethanol. The product obtained is subjected to furtherchromatography and recrystallization, which resulted in a pure qualityof product good for injection production.

[0012] Purification of the crude product containing the mixture ofcyclosporins is very difficult, since the impurities having similarchemical structures are very similar in chromatographic characteristicsto cyclosporin A as the main product. As the previously describedprocesses prove, that regardless of the employed solvent mixture andbecause of the overlapping of the chromatographic peaks furtherchromatographic or other purification steps have to be carried out forobtaining certain components in pure form. The purification processesknown so far are generally characterized by not only the fact, thattheir solvent requirement is considerable large, but the application of3-4 different type of solvents or solvent mixtures and 2-3 types ofcolumn fillings are needed as well. As a consequence of these factsseveral type of chromatographic technics and regeneration methods arerequired within a process, which make difficulties in the development ofa simple constructed and uniformly manageable economic industrial scaletechnology.

[0013] From environment protection point of view further problems areraised by the application of chlorinated hydrocarbons, since more andmore countries have been making efforts for the limitation of theirusage.

[0014] Regarding the filling materials of columns the application ofaluminum oxide filling for industrial purposes is very doubtful, becauseas a consequence of its small specific surface the loading capacity andthe separation ability is very small. Furthermore it is not advantageousfor industrial purposes, because the aluminum oxide filling is rigid andeasily fragile, thus it needs a special equipment and technology. Thosecan not be used in frequently emptied stainless steel columns generallyused in the chemical industry.

[0015] Sephadex type fillings are very expensive ones, and in the caseof the cyclosporin complex their efficiencies are very limited, sincethe sizes of molecules are very near to each others.

[0016] The object of the present invention is the development of aneasily applicable chromatographic purification method in industrialscale which is suitable to manufacture the cyclosporin A ingredientcontaining much less impurities so far thus using it in medical practicein a safer way.

[0017] Our aim was to develop such a chromatographic purificationtechnology, which requires only one type of solvent mixture and one typeof column filling.

[0018] Because of its advantageous characteristics—high specificsurface, high pore size, favorable sorption ability, easy handling andrelatively low price—silica gel was used as a column filling. An idealsolvent mixture and a method had to be selected to this filling which issuitable to separate the cyclosporin components with high selectivity.

[0019] As a result of our experiments we recognized that our aim can berealized by multistep chromatography on silica gel column using asolvent mixture as an eluent which main component is toluene.Surprisingly it was found that even cyclosporin U and L components beingthe nearest to cyclosporin A can be separated by a three stageschromatography on silica gel column using toluene containing acetone asan eluent. These components differ from cyclosporin A only in a methylgroup.

[0020] According to the first example of the application No. WO 94/16091a solvent mixture of toluene and acetone is also used in a single stage.The product obtained is recrystallized from a solvent mixture of etherand hexane. (Yield: 66.6%.) The optical rotation of the product issufficient, but its melting point is remarkably lower than thatdescribed in the literature, which indicates that the product is notpure. Thus, even recrystallization from a solvent will yield product ofpoor purity and a low yield.

[0021] Process according to the present invention is suitable toseparate the most frequent impurities such as cyclosporins B and C beingat present the largest quantities and moreover cyclosporin D, U and Lcomponents being at present in trace quantities. Cyclosporins B and Ccontent of cyclosporin A end product obtained this way is less then 0.02wt %, while cyclosporins L, U and D content is below 0.05 wt %.

[0022] The object of our invention is an improved process for thepurification of cyclosporin A from a crude product containingcyclosporin complex which also enables the production of extremely purecyclosporin A in large scales, with a chromatographic method on silicagel column using multistep chromatography with a solvent mixturecontaining toluene as the main component. Another new feature of theprocess is that we apply extremely high column load. In the conventionalchromatographic practice the extent of column load amounts to not morethan 5-10% of the column charge, and this value is lower forcyclosporins.

[0023] The crude product as used herein, refers to a product isolatedfrom a fermentation broth by an extraction process. The crude productmay contain, in addition to a cyclosporin complex, other organic orinorganic components. A cyclosporin complex, as used herein, is acomplex which includes cyclosporin A and at least one other cyclosporin.

[0024] The chromatographic method of the procedure employs a stationaryphase and a mobile phase. In the preferred mode, designated herein asmultistep chromatography, the stationary phase consists of multiplenormal phase columns. The normal phase column is typically a silica gelcolumn or a material with equivalent properties. The mobile phasesconsists of a solvent or a solvent mixture which in the preferred modecontains toluene as the main component.

[0025] A new feature of the process is that we apply extremely highcolumn load at least at the first purification step. Or preferably, weapply extremely high column load at least at one purification step.Column load refers to the cyclosporin A component of column charge, thatis the mass of cyclosporin A loaded onto the first column relative tothe mass of silica gel. In the conventional chromatographic practice theextent of column load amounts to not more than 5-10% of the columncharge, and this value is lower for cyclosporins. In the presentinvention, the total mass of compounds applied to the first column isgreater than about 20% of the mass of the silica gel absorbent. Morepreferably it is between, about 20 and 40% and most preferably betweenabout 25 and 35%, of the mass of the silica gel absorbent. The extremelyhigh column load of the present invention will accommodate a mass ofcyclosporin A greater than about 0.100 kg, preferably between about0.100 to 5.0 kg, more preferably between about 2 -4.5 kg in the case ofan 8 liter column. Higher column volumes causes a proportional mass ofcyclosporin A. Thus, the larger the column volume, the larger the massof cyclosporin A that may be purified by the methods of the presentinvention. For example, by the process of the present invention it ispossible to purify even 10-20 kg samples of cyclosporin A using anextremely high column load and using a column that has an appropriatevolume.

[0026] The multistep chromatography, the solvent mixture containingtoluene as the main component and the high column load are interrelatedand the desired result can only be achieved by their combined use.Therefore all three of above properties should be used simultaneously soas to obtain the extremely pure product.

[0027] According to the present invention preferably 2-4 chromatographicsteps are applied subsequently and more expediently three steps.

[0028] The overload of the column is the highest in the first step ofchromatography. The definite separation of the active substance and theimpurities allows the use of high column load in the subsequent twosteps of chromatography as well.

[0029] For the purification it is favorable to use a solvent mixture oftoluene-acetone, which contains at most 30 vol % acetone.

[0030] According to all other favorable method a solvent mixture oftoluene-ethyl acetate is used in which the ethyl acetate concentrationis below 35 vol %.

[0031] In the purification process at least one time it is favorable touse gradient elution.

[0032] According to our experiments it was found that for thepurification of the cyclosporin complex processed in our case isfavorable to use 10-30 vol % and more favorable to use 13-18 vol %acetone and 10-35 vol % or 15-20 vol % ethyl acetate.

[0033] According to a possible method of the present invention theelution is done in the case of the three steps chromatography withtoluene containing 15 vol % acetone or 18 vol % ethyl acetate. In thefirst step the major part of cyclosporin C is separated, in the secondstep the larger part of cyclosporin B, L and U components are removed,and finally in the third step the quantities of L and U components andother unidentified impurities can be reduced below 0.05 wt %. In thefirst step the cyclosporin A loss is minimal, however in thepre-fractions of the second and third steps a considerable quantity ofcyclosporin A is removed together with the cyclosporin D component beingvery close to cyclosporin A. This cyclosporin A can be recovered in avery pure form in the fourth step.

[0034] For comparison in the table below the impurity profiles ofcyclosporin A USP standard, the cyclosporin A active ingredient fromSANDIMMUN® injection and also the data of the cyclosporin A productprepared by the present invention are given. Product according to USPstandard SANDIMMUN ® inj. the Example 1 Impurities wt % wt % wt %Cyclosporin C <0.02 0.17 0.05 Cyclosporin B <0.02 0.21 0.05 Unknown 0.090.35 0.05 Cyclosporin L 0.05 0.35 0.05 Cyclosporin U — — 0.05Cyclosporin D 0.12 — 0.05

[0035] From the data it can be seen, that the quality of the cyclosporinA obtained by the present invention considerably exceeds the parametersof the SANDIMMUN® injection, as well as the USP requirements too.

[0036] Process according to the present invention is applicable for thepurification of crude product both in small and large scales too.

[0037] Beside the fact that the process according to the presentinvention is able to prepare pure cyclosporin A, it has some notnegligible advantages too. Since only one type of technological method(chromatography) is used, the purification process is manageableuniformly, moreover, it is repeatable and can be converted into acontinuous process. Furthermore, there is a special advantage, thatsince only one type of solvent mixture is used in the threechromatographic steps, the regeneration of both the columns and thesolvents became more simple.

[0038] A further special benefit of the process is that the first stepof chromatography the highly binding impurities remain on the column,which greatly facilitates the regeneration of column charges in thesubsequent two steps.

[0039] The present invention is introduced by the examples below withoutlimiting the protection demand only to them.

[0040] In a comparative example (Example 4) it is presented, that withthe application of a solvent mixture of dichloromethane-acetone in 3steps chromatography generally used so far in the known process, couldnot be produced a final product in a similar purity.

EXAMPLE 1 Purification of Cyclosporin Crude Product with Three StepsChromatography with the Application of a Solvent Mixture ofToluene-Acetone

[0041] Quality of the starting cyclosporin crude product:

[0042] Cyclosporin A content 60.9 wt %

[0043] Cyclosporin B content 11.2 wt %

[0044] Cyclosporin C content 8.3 wt %

[0045] Cyclosporin L content 1.79 wt %

[0046] Cyclosporin U content 1.58 wt %

[0047] Cyclosporin D content 1.25 wt %

[0048] 1st Step

[0049] Chromatography is done with two chromatographic columns connectedin series, each one is 8 literss with jacket, diameter 10 cm, length 100cm. Each of the two columns contains 3.95 kg Merck type Kieselgel silicagel with grain size 0.04-0.063 mm. In the First chromatography bothcolumns contain fresh silica gel. In the case of the next chromatographythe first column is separated and a second column is connected to itcontaining fresh silica gel. Further on for each chromatography only onenew column is used. When the columns are changed and we use columnsfilled with fresh and used silica gel the column load is preferablybetween about 25% and about 80% calculated on the fresh silica gel.

[0050] Preparation of the Crude Product

[0051] 4.1 kg with a purity of 60.9 wt % crude product is loaded to apair of columns connected in series being dissolved previously in 15liters toluene. 19 liter solution is received which is subjected to thetop of the first column through a filter with a 2.4 l/h feeding rate.After loading the material is eluted with a solvent mixture of 13:87 vol% acetone-toluene until the volume of the effluent at the bottom of thesecond column is 39 liters. The cyclosporin content of the effluent isanalyzed by TLC. Fractions not containing cyclosporin are collected aswaste. 28 liter of the effluent after appearing the cyclosporin isconsidered as the main fraction. Dry material content of theintermediate I obtained this way is 3.23 kg.

[0052] Quality:

[0053] cyclosporin A 75 wt %

[0054] cyclosporin B 10. 1 wt %

[0055] cyclosporin C 1.6 wt %

[0056] cyclosporin L 1.7 wt %

[0057] cyclosporin U 1.5 wt %

[0058] cyclosporin D 1.3 wt %

[0059] Yield calculated to cyclosporin A: 97%

[0060] 2^(nd) Step

[0061] Separation is done in a 1 meter length jacketed 8 liter Column.Column contains Merck Kieselgel 60 silica gel (0.015-0.040 mm). Mass ofthe filling is 3.95 kg. About 3 liter volume and 370 g dry materialcontaining intermediate I solution obtained in the first step issubjected to the column with 2.4 l/h feeding rate, then it is washedwith 1 liter toluene.

[0062] After loading the material the column is eluted with the mixturesof 10 liter 15:85 vol % acetone:toluene and then with 20 liter 25:75 vol% acetone:toluene solutions. The flow rate of the solvent till 17 literfraction is 2.4 l/h, then from 18 liter fraction 5 l/h. Fractions areanalyzed by TLC. 1 -11 liter fractions are waste, fractions 12-19 literare considered as critical 1 fractions and samples are taken from them.Impurity profiles of them are analyzed by FLPLC and those are handled aspre-fractions or combined with the main fractions. This way 80 g drymaterial containing pre-fraction can be received which is thenevaporated to dryness.

[0063] 20-25 liter fractions are combined as main fraction. 26-31 literfractions are considered as critical fractions and after analysis thoseare combined with the main fraction or handled as post-fraction.

[0064] Main fraction obtained the above way is evaporated to dryness ina film evaporator mounted with an oscillating stirrer. 234 g ofintermediate 11 is obtained with a yield of 80% and with a quality ofthe following:

[0065] cyclosporin A 95 wt %

[0066] cyclosporin U 1.2 wt %

[0067] cyclosporin L 0.7 wt %

[0068] cyclosporin B<0.1 wt %

[0069] cyclosporin D 0.5 wt %

[0070] cyclosporin C 0.1 wt %

[0071] 3^(rd) Step

[0072] Chromatography is done on the columns with the same constructionand geometric sizes described in the 1st and 2nd steps.

[0073] A 1.7 liter toluene solution containing 220 g dry material isprepared from the intermediate 11 obtained in the second step andsubjected to the top of the column with 2.4 l/h feeding rate, then it iswashed with 1 liter toluene.

[0074] The column is eluted with a mixture of 20 liter 15:85 vol %acetone:toluene and then the cyclosporin is eluted with a mixture of 20liter 25:75 vol % acetone:toluene.

[0075] The flow rate of the elution till 31 liter fraction is 2.4 l/h,then from 32 liter fraction 5 l/h.

[0076] 1 -18 liter fractions are waste, fractions 19-23 liter arepre-fractions and considered as critical I fractions. Samples are takenfrom them in order to analyze the dry material content and the impurityprofiles by HPLC. After analysis those are handled as pre-fractions orcombined with the main fractions. 29-38 liter fractions are combined asmain fraction. 39-41 liter fractions are collected in 1 liter portionsand considered as critical fractions 11. After analysis those arecombined with the main fraction or handled as post-fraction.

[0077] As a result of the fractions collection after evaporation todryness 70 g pre-fraction can be received.

[0078] Main fractions are combined and after evaporation to dryness 157g pure cyclosporin is obtained with a yield of 75%. Quality of theproduct is the following:

[0079] cyclosporin A 99.6 wt %

[0080] cyclosporin L<0.05 wt %

[0081] cyclosporin U<0.05 wt %

[0082] cyclosporin D<0.05 wt %

[0083] cyclosporin B<0.02 wt %

[0084] cyclosporin C<0.02 wt %

EXAMPLE 2 Chromatographic Purification of Cyclosporin Crude Product onStationary Bed Four Steps Silica Gel Column with the Application ofSolvent Mixtures of Toluene-acetone or Toluene-ethyl Acetate

[0085] Cyclosporin Crude product is purified with a three stepschromatography described in Example 1. Pre-fractions received in thethree steps chromatography are purified in the fourth step in stationarybed with a solvent mixture of toluene-ethyl acetate.

[0086] 4^(th) Step

[0087] Construction and geometric sizes of chromatographic column is thesame as described in Example 1. Column contains Merck Kieselgel 60 typesilica gel (0.015-0.040 mm) as it is written in Example 1.

[0088] Column is subjected by a concentrate received from pre-fractionin the quantity of 260 g material dissolved in 2.5 liter toluene with afeeding rate of 2.4 l/h.

[0089] cyclosporin A content 80.6 wt %

[0090] cyclosporin D content 4.2 wt %

[0091] Subjected sample is washed with 1 liter toluene, then the columnis eluted with a mixture of 20 liter 17:83 vol % ethyl acetate:toluene,by a flow rate of 2.4 l/h, then the elution is continued with a mixtureof 40 liter 28:72 vol % ethyl acetate:toluene.

[0092] In the course of fractions collection the 1-19 liter fractionsare waste. After HPLC analysis the 20-25 liter fractions are waste orcombined with the main fraction. 26-35 liter fractions are collected asmain fraction. After sampling and HPLC analysis the 36-42 literfractions either combined with the main fraction or handed as waste.23-42 liter collected main fraction described above is evaporated todryness. This way 195 g pure cyclosporin A is obtained containing 99.6wt% active ingredient with a yield of 75% and with a quality of thefollowing:

[0093] cyclosporin A 99.6 wt %

[0094] cyclosporin D<0.05 wt %

[0095] cyclosporin U -

[0096] cyclosporin L -

EXAMPLE 3 Chromatographic Purification of Cyclosporin Crude Product onStationary Bed Two Steps Silica Gel Column with the Application of aSolvent Mixture of Toluene-acetone

[0097] Cyclosporin crude product is purified according to the sameprocess described in the 1st step of Example 1 obtaining cyclosporinintermediate I with the same quality. Further on the process is thefollowing:

[0098] 2^(nd) Step

[0099] Column is subjected by 3 liter intermediate1 containing 370 gdried material with a feeding rate of 2.4 l/h, then the subjected sampleis washed with 1 liter toluene.

[0100] After loading the column is eluted with a mixture of 10 liter15:85 vol % acetone:toluene, then the elution is continued with amixture of 20 liter 25:75 vol % acetone:toluene.

[0101] The flow rate of the solvent is 2.4 l/h till 17 liter and 5 l/hfrom 18 liter.

[0102] According to TLC and HPLC detection 1-11 liter fractions arewaste, 12-20 fractions are pre-fractions, 21-24 fractions are consideredas the main fractions and fractions from 25 till the end are handled aspost-fractions. Washing acetone fractions are waste.

[0103] After fractionation, the collected main fractions are combinedand evaporated to dryness obtaining 114 g cyclosporin A with a yield of41% and with as good quality of product as is written in Example 1.

EXAMPLE 4 Comparative Example for the Purification of Crude Product with3 Steps Chromatography with the Application of a Solvent Mixture ofDichloromethane-acetone

[0104] Quality of the starting cyclosporin crude product (the same asused in Example 1):

[0105] Cyclosporin A content 60.9 wt %

[0106] Cyclosporin B content 11.2 wt %

[0107] Cyclosporin C content 8.3 wt %

[0108] Cyclosporin L content 1.79 wt %

[0109] Cyclosporin U content 58 wt %

[0110] Cyclosporin D content 1.25 wt %

[0111] 1^(st) Step

[0112] Chromatographic equipment and the filling is the same asdescribed in Example 1. One pair of column connected in series issubjected with 4.1 kg crude product with the purity of 60.9% in 15 literdichloromethane solution.

[0113] After loading the sample the column is eluted withdichloromethane with a flow rate of 2.4 l/h till collecting 35 liter ofeffluent.

[0114] 1-10 liter fractions are waste, while 11-35 liter fractions areconsidered as main fractions. Dried material content of intermediate 1obtained this way is 2.9 kg, active ingredient content is 75 wt %.

[0115] 2^(nd) Step

[0116] Chromatographic equipment and the filling is the same asdescribed in Example 1.

[0117] Column is subjected at the top by 3 liter intermediate 1containing 350 g dried material with a feeding rate of 2.4 L/h. Elutionis done with a mixture of 10 liters acetone:dichloromethane 1:9 vol.rate, then continued with a mixture of 25 liter acetone:dichloromethane2:8 vol, rate and finished with acetone 2.4 l/h rate.

[0118] According to TLC analysis the volume of the pre-fraction was 13liters, the volume of the main fraction was 22 liter, and the volume ofthe post-fraction was 11 liter. 22 liter of main fraction was evaporatedto dryness and as a consequence 220 g intermediate 11 product wasobtained with the purity of 91%.

[0119] 3^(rd) Step

[0120] Chromatographic equipment and the filling is the same asdescribed in Example 1.

[0121] Filling is loaded by 220 g intermediate 11 feeding thedichloromethane concentrate with a rate of 2.4 l/h on the column.Elution is done with a mixture of 20 liters acetone: dichloromethane 1:9vol. rate, then continued with a mixture of 30 liter acetone:dichloromethane 2:8 vol. rate and finished with acetone 2.4 l/h rate.

[0122] First 26 liter fractions are considered as pre-fractions, then 26liter main fraction is collected and finally 11 liter post-fractions aretaken.26 liter of main fraction was evaporated to dryness and as aconsequence 140 g intermediate III product was obtained with thefollowing quality:

[0123] Cyclosporin A 98.6 wt %

[0124] Cyclosporin U 0.6 wt %

[0125] Cyclosporin D 0.3 wt %

[0126] Cyclosporin L 0.2 wt %

[0127] Cyclosporin B 0.1 wt %

[0128] Cyclosporin C 0.1 wt %

[0129] Although certain presently preferred embodiments of the inventionhave been described herein, it will be apparent to those skilled in theart to which the invention pertains that variations and modifications ofthe described embodiment may be made without departing from the spiritand scope of the invention. Accordingly, it is intended that theinvention be limited only to the extent required by the appended claimsand the applicable rules of law.

1. A purification process for the preparation of cyclosporin A in highpurity from crude product containing cyclosporin complex comprisingmultistep silica gel column chromatography in which the mobile phase isa solvent comprising toluene as the main component.
 2. The process ofclaim 1, consisting of 2-4 chromatographic steps.
 3. The process ofclaim 1, consisting of 3 chromatographic steps.
 4. The process of claim1, wherein toluene-acetone is used as a solvent.
 5. The process of claim4, wherein toluene containing at most 30 vol % acetone is used.
 6. Theprocess of claim 1, wherein a solvent of toluene-ethyl acetate is used.7. The process of claim 6, wherein toluene containing at most 35 vol %ethyl acetate is used.
 8. The process of claim 1, wherein gradientelution is used in the case of at least one chromatographic step.
 9. Theprocess of claim 1, wherein the cyclosporin A has a mass of greater thanabout 20% of the silica gel mass.
 10. The process of claim 1, whereinthe cyclosporin A has a mass of between 25% and 35% of the silica gelmass.
 11. The process of claim 1, wherein the cyclosporin A loaded ontothe silica gel column has a mass greater than about 0.1 kg.
 12. Theprocess of claim 1, wherein the cyclosporin A loaded onto the column hasa mass between about 0.100 to 5.0 kg.
 13. The process of claim 12wherein the column has a volume of about 8 liters.
 14. The process ofclaim 1, wherein the cyclosporin A loaded onto the column has a massbetween about 2 to 4.5 kg.
 15. The process of claim 14 wherein thecolumn has a volume of about 8 liters.
 16. An industrial scale processfor the purification of cyclosporin A from cyclosporin complexcontaining crude product comprising multistep silica gel columnchromatography using normal phase silica gel as a stationary phase and asolvent comprising toluene as the main component of the mobile phase.17. The process of claims 1 or 16, wherein the cyclosporin complex usedas the starting material of the chromatography is heated to 80-120° C.prior to chromatography.
 18. The process of claims 1 or 16, whereintoluene containing 10-30 vol % acetone is used.
 19. The process ofclaims 1 or 16, wherein toluene containing 10-35 vol % ethyl acetate isused.
 20. The process of claim 16, wherein the cyclosporin A has a massgreater than about 20% of the silica gel mass.
 21. The process of claim16, wherein the cyclosporin A has a mass between about 25% and 35% ofthe silica gel mass.
 22. The process of claim 21 wherein thechromatography is performed using a column with a volume of about 8liters.
 23. The process of claim 16 wherein the mass of cyclosporin Aloaded onto the column is greater than about 0.1 kg.
 24. The process ofclaim 16 wherein the mass of cyclosporin A loaded onto the column isbetween about about 0.100 to 5.0 kg.
 25. The process of claim 24 whereinthe chromatography is performed using a column with a volume of about 8liters.
 26. The process of claim 16 wherein the mass of cyclosporin Aloaded onto the column is between about 2 to 4.5 kg.
 27. The process ofclaim 26 wherein the chromatography is performed using a column with avolume of about 8 liters.